The goal of this research is to identify immunological and developmental barriers that limit interspecies chimera formation, with the long-term aim of identifying robust and ethically-acceptable approaches to generate human organs for clinical transplantation. Generation of human organs on-demand would compensate for the chronic shortage of organ donors. However, in vitro generation of whole organs has proved difficult. To address this issue, my laboratory has demonstrated proof-of-concept for generating functional organs in vivo by repurposing the ?developmental niche? of a host species to grow an organ of a second (donor) species. After injecting mouse pluripotent stem cells (PSCs) into Pdx1-/- (pancreatogenesis-deficient) rat blastocysts, the mouse cells complemented the empty organ niche and developed a mouse pancreas in a rat. Highlighting the therapeutic potential of this approach, islets prepared from this mouse pancreas could maintain normal blood glucose levels in diabetic mice following transplantation. Transplanting such genetically-matched organs would bypass the detrimental requirement for life-long immunosuppression. While interspecies organogenesis has the potential to solve the organ-donor shortage, several ethical and practical issues must be addressed before we can move to studies involving human organogenesis in animals. In this proposal, we aim to tackle two key issues by studying interspecies rodent chimeras. The first issue is that donor engraftment rates in interspecies chimeras are often low, regionally variable, and lead to embryonic lethality. We hypothesize that the existence of a xenogenic barrier limits interspecies engraftment and development. Loss of donor engraftment often occurs concomitantly with formation of the immune system, suggesting an immunological component of the xenobarrier. By identifying and modulating constituents of this xenobarrier, we aim to develop strategies to improve interspecies organ generation. The second issue is an ethical concern regarding generation of systemic chimeras in which human PSCs could contribute to the brain (ectoderm) or germ cells of other animals. We hypothesize that these current roadblocks can be overcome by using lineage-committed progenitor cells that have lost the developmental potential to generate brain or germ cells. Additionally, limiting chimerism to a specific region may overcome the lethal effects of systemic engraftment while allowing high levels of regional chimerism. My laboratory has already demonstrated proof-of-concept for the use of lineage-committed endodermal progenitors in an intraspecies context. Endodermal progenitors can only contribute to endodermal tissues (e.g. pancreas), affording a ?targeted? chimerism approach. Here, we will explore the plasticity and limits of heterochronic progenitor engraftment and aim to validate interspecies targeted organogenesis in mouse-rat and human-rat chimeras. In summary, by applying the above advances to investigate the biology of interspecies organogenesis, we will develop robust strategies for ethically-acceptable human organ generation.